Bax transmembrane domain interacts with prosurvivalBcl-2 proteins in biological membranesVicente Andreu-Fernándeza,b,c, Mónica Sanchoa, Ainhoa Genovésa, Estefanía Lucendoa, Franziska Todtb,Joachim Lauterwasserb,d, Kathrin Funkb,d, Günther Jahreise, Enrique Pérez-Payáa,f,1, Ismael Mingarroc,2, Frank Edlichb,g,2,and Mar Orzáeza,2

aLaboratory of Peptide and Protein Chemistry, Centro de Investigación Príncipe Felipe, E-46012 Valencia, Spain; bInstitute for Biochemistry and MolecularBiology, University of Freiburg, 79104 Freiburg, Germany; cDepartament de Bioquímica i Biologia Molecular, Estructura de Recerca Interdisciplinar enBiotecnología i Biomedicina, Universitat de València, 46100 Burjassot, Spain; dFaculty of Biology, University of Freiburg, 79104 Freiburg, Germany;eDepartment of Biochemistry/Biotechnology, Martin Luther University Halle-Wittenberg, 06120 Halle, Germany; fInstituto de Biomedicina de Valencia,Instituto de Biomedicina de Valencia–Consejo Superior de Investigaciones Científicas, 46010 Valencia, Spain; and gBIOSS, Centre for Biological SignalingStudies, University of Freiburg, 79104 Freiburg, Germany

Edited by William F. DeGrado, School of Pharmacy, University of California, San Francisco, CA, and approved November 29, 2016 (received for review July28, 2016)

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X,apoptosis regulator) can commit cells to apoptosis via outer mito-chondrial membrane permeabilization. Bax activity is controlled inhealthy cells by prosurvival Bcl-2 proteins. C-terminal Bax trans-membrane domain interactions were implicated recently in Bax poreformation. Here, we show that the isolated transmembrane domainsof Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediateinteractions between Bax and prosurvival proteins inside the mem-brane in the absence of apoptotic stimuli. Bcl-2 protein transmem-brane domains specifically homooligomerize and heterooligomerizein bacterial and mitochondrial membranes. Their interactions partic-ipate in the regulation of Bcl-2 proteins, thus modulating apoptoticactivity. Our results suggest that interactions between the trans-membrane domains of Bax and antiapoptotic Bcl-2 proteins repre-sent a previously unappreciated level of apoptosis regulation.

apoptosis | Bcl-2 | mitochondria | oligomerization | transmembrane

The mitochondrial apoptosis program can activate the proa-poptotic Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 as-

sociated X, apoptosis regulator) in response to stress, resulting inouter mitochondrial membrane (OMM) permeabilization and therelease of cytochrome c (cyt c) and other proteins of theintermembrane space into the cytosol. The Bcl-2 family controls Baxactivity and thus the integrity of the OMM (1–3). Prosurvival Bcl-2proteins harbor four Bcl-2 homology domains [BH1–4, as repre-sented by Bcl-2, Bcl-xL (B-cell lymphoma-extra large), or Mcl-1] andcounteract proapoptotic Bcl-2 proteins with three BH domains(BH1–3; e.g., Bax or Bak). The diverse group of BH3-only proteinsregulates both prosurvival and proapoptotic Bcl-2 proteins. Pro-survival Bcl-2 proteins either inhibit Bax via direct interaction or bysequestering “activator” BH3-only proteins, thus preventing theirinteraction with Bax (4–9).In healthy cells, newly synthesized Bax initially translocates to

the OMM, but efficiently retrotranslocates to the cytoplasm,depending on prosurvival Bcl-2 proteins (10, 11). Bax shuttling thusestablishes an equilibrium between cytosolic and mitochondriallyanchored molecules (10, 12), determining the cellular response toapoptotic stress (13). Upon the induction of apoptosis, Bax andBak interact and at least partially insert into the OMM (14–16).Regulatory interactions between Bax and other Bcl-2 proteins canonly be observed in the presence of the OMM or liposomes (17,18). Recent studies suggest that Bax is inserted in mitochondrialmembranes as a monomer that oligomerizes once inserted (19–21).These studies also have shown that Bcl-xL inhibits Bax by dissoci-ating Bax oligomers. However, the contribution of the differentprotein domains to oligomer formation and apoptosis modulationwithin the membrane is still unclear.Transmembrane domains (TMDs) can mediate protein–protein

interactions within membranes and be involved in signal transduction

across bilayers via changes in the oligomeric state or protein con-formation (22–24). The Bax TMD targets fusion proteins to theOMM; its deletion results in cytosolic Bax localization and impairedBax activity (25). Analysis of the active Bax membrane topologysuggests that the TMD could play a central role in Bax oligomeri-zation (26). Förster resonance energy transfer studies have shownthat Bax forms homooligomers in the mitochondria through TMDinteractions (27). Bcl-xL–mediated Bax retrotranslocation into thecytosol depends on the Bcl-xL TMD, suggesting the involvement ofTMD interactions in Bax inhibition (13). In addition, distancemapping of cysteine-labeled Bax variants in large unilamellar vesiclessuggests a role of the Bax TMD in the formation of potential Baxpore structures (28). Thus, TMD interactions could be involved inBax regulation, oligomerization, and pore formation.Here, we report the self-association and interaction of the TMDs

of Bax, Bcl-2, and Bcl-xL in the biological membranes of living cellsin the absence and presence of apoptosis induction. The TMDsmediate homooligomerization and heterooligomerization betweenproapoptotic Bax, and prosurvival Bcl-2 and Bcl-xL members in-dependent of extramembrane protein regions, modulating the re-sponse to apoptosis signaling.

Significance

Bcl-2 (B-cell lymphoma 2) proteins are key regulators of apo-ptosis. The recruitment of the predominantly cytosolic Bcl-2protein Bax (Bcl-2 associated X, apoptosis regulator) to themitochondria is associated with mitochondrial outer mem-brane permeabilization and apoptosis. We report specific in-teractions between the transmembrane domains (TMDs) of Baxand the prosurvival Bcl-xL (B-cell lymphoma-extra large) andBcl-2 proteins. Our results demonstrate that these interactionsoccur in nonapoptotic human cells and participate in the reg-ulation of Bcl-2 proteins, introducing the concept of modula-tion mitochondrial apoptosis signaling by TMD-mediated Bcl-2protein interactions.

Author contributions: V.A.-F., M.S., A.G., E.P.-P., I.M., F.E., and M.O. designed research;V.A.-F., M.S., A.G., E.L., F.T., J.L., G.J., and M.O. performed research; K.F. and M.O.contributed new reagents/analytic tools; I.M., F.E., and M.O. analyzed data; and I.M.,F.E., and M.O. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1Deceased May 28, 2013.2To whom correspondence may be addressed. Email: morzaez@cipf.es, Ismael.Mingarro@uv.es, or frank.edlich@biochemie.uni-freiburg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1612322114/-/DCSupplemental.

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Results and DiscussionBax, Bcl-xL, and Bcl-2 TMDs Homooligomerize in E. coli Membranes.Interactions between Bcl-2 proteins require membranes (29, 30).Therefore, the potential homooligomerization of Bax, Bcl-2, andBcl-xL TMDs was analyzed in living cells by using the ToxRedsystem (31). In this assay, TMDs were inserted between thetranscriptional activation domain (ToxR) and maltose-bindingprotein (MBP), targeted to the periplasm (32). Transcriptionalactivation of the cholera toxin promoter strictly depends onToxR oligomerization (33). Consequently, TMD oligomerizationresulted in red fluorescent protein (RFP) expression (Fig. 1A).The level of fluorescence is proportional to the analyzed oligo-merization. Fusion protein functionality, orientation, and mem-brane insertion were controlled by the growth of mutantEscherichia coli on maltose as the sole carbon source dependenton MBP activity in the periplasm (Fig. S1). The TMDs of Bcl-xLand Bcl-2 induced as much RFP fluorescence and thereforeoligomerization as the positive control, glycophorin A (GpA)TMD (Fig. 1B). Similar levels of Bax TMD generated the highestfluorescence levels (Fig. 1B). The emission spectra of ToxRedchimeras corroborate the formation of Bcl-2 protein homo-oligomers (Fig. S2). Interestingly, the amino acid sequences ofthe Bax, Bcl-xL, and Bcl-2 TMDs (Table 1) revealed centralglycine residues as potential sites for strong helix–helix interac-tions through ridge-into-grove arrangements, as observed forother interacting TMDs (24, 34–37). These glycine residues are evo-lutionarily conserved (Fig. S3). Structural studies have demonstrated

that conserved glycine residues rarely face lipids, and many of themparticipate in close helix–helix packing (38). Accordingly, Fig. 1Cshows disruptive effects for mutations Bcl-2 G227I, Bcl-xL G222I,and, to a lesser extent, for Bax G179I (similar to the GpA G83Imutant) (39). The helix-breaking G179P mutation [similar to GpA(40)] resulted in an even-more-prominent disruption of Bax TMDhomooligomerization, whereas mutation of the conserved BaxF176 had no effect. In all cases, expression levels, as determinedby Western blotting, were comparable (Fig. 1 B and C). Fur-thermore, appropriate membrane insertion of chimeric proteinswas confirmed (Fig. S4). Therefore, the TMDs of Bax, Bcl-2, andBcl-xL mediate specific and efficient homooligomerization ina biological membrane.

Bax, Bcl-xL, and Bcl-2 TMDs Form Oligomers in Mitochondrial Membranes.Next, we analyzed the self-association capacity of Bax, Bcl-xL, andBcl-2 TMDs in nonapoptotic human cells using bimolecular fluo-rescence complementation (BiFC) assays (41). TMDs were fusedwith two nonfluorescent fragments (VN: 1–155 amino acids, I152L;and VC: 155–238 amino acids, A206K) of the venus fluorescentprotein to assess whether TMD interactions would reconstitutefluorescence of venus (Fig. 2A) (42). VN and VC constructs fused tothe same TMDwere cotransfected in HCT116 colon carcinoma cells.This process, indeed, resulted in Bax, Bcl-2, and Bcl-xL TMDhomooligomerization in the absence of apoptotic stimuli (Fig. 2B).The nonoligomerizing TMD of the abundant mitochondrial Tom20protein was used as a negative control for overcrowding. Bcl-xLoligomerization is still controversial; some cross-linking experimentssuggest oligomer formation, whereas other studies using detergents orstructural data in nanodiscs have revealed monomers (43). However,extraction of transmembrane proteins with detergents could dissoci-ate them, and nanodisc studies are highly dependent on the lipidcomposition (44). A striking advantage of the BiFC assays is theobservation of interactions in eukaryotic membranes. The Westernblot analysis confirmed comparable expression levels of the VN andVC constructs. Therefore, the observed fluorescence values indicatedifferent association levels and agree with the results obtainedin the ToxRed system (Fig. 1B and Fig. S5), where self-association ofthe proapoptotic Bax TMD caused the highest fluorescence signal.The analysis of the intracellular distribution of TMD fusion proteinsby confocal microscopy revealed mitochondrial localization (Fig. 2C).Next, single amino-acid substitutions were introduced into the

TMDs to analyze the sequence specificity of TMD homooligome-rization (Fig. 2D). The subcellular distribution of chimeric con-structs was also corroborated by cellular fractionation (Fig. 2E). Inagreement with ToxRed homooligomerization experiments, stronginterference by Bax G179P (see also Fig. 2C, bottom row), Bcl-2G227I, and Bcl-xL G222I was observed, whereas Bax F176A againexhibited no difference from the wild-type construct. The Bcl-2L225P and Bcl-xL G217P mutants also exhibited strong interferencewith TMD oligomerization. Therefore, Bax, Bcl-2, and Bcl-xLTMDs form specific homooligomers within the OMM.

Bax TMD Interacts with Prosurvival Bcl-2 Protein TMDs. Because ofthe specific homooligomerization of the Bax, Bcl-2, and Bcl-xLTMDs in nonapoptotic cells, the possibility of heterooligomeriza-tion and regulatory interactions between these TMDs was tested.

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Fig. 1. Bax, Bcl-xL, and Bcl-2 TMDs homooligomerize in membranes. (A) TMDconstructs fused to ToxR were expressed in the inner membrane of E. coli. TheTMD interaction reconstituted active ToxR and activated ctx promoter-regulated RFP expression. The C-terminal MBP domain was exposed to theperiplasm. (B) ToxR-TMD-MBP constructs (Table 1) were transformed intoMM39 cells. The fluorescence signal (ʎexc 570, ʎem 620 nm) obtained with theToxRed system for Bax, Bcl-xL, and Bcl-2 TMDs is shown. Red bars highlight in-teractions. The GpA TMD and nondimerizing TMD GpA G83I variant served aspositive and negative controls, respectively. Western blotting of MBP revealedequivalent expression levels. (C) Homooligomerization assay of wild-type andvariants of the Bax, Bcl-xL, and Bcl-2 TMDs. Fusion protein functionality, orien-tation, and membrane insertion of all variants were controlled by growth onminimum medium (Fig. S4). Reduction in fluorescence indicates the disruptionof homooligomerization (white bars). Results obtained with mutations thatapparently do not interfere with homooligomerization are depicted in brown.Error bars represent the mean ± SD; n ≥ 3. Western blotting of MBP revealedequivalent expression levels for all chimeras. P values according to Dunnett’stest are displayed. *P < 0.05; **P < 0.01; ***P < 0.001.

Table 1. Sequences of the TMDs of human Bax, Bcl-xL, andBcl-2 proteins

Protein Accession no. Amino acid sequence

Bcl-xL NP_612815 210FNRWFLTGMTVAGVVLLGSLFSR232

Bcl-2 NP_000624 214WLSLKTLLSLALVGACITLGAYL236

Bax Q07812 169TWQTVTIFVAGVLTASLTIW188

Central glycine residues are shown in bold, and mutated Phe176 from Baxis in italics.

2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1612322114 Andreu-Fernández et al.

To this end, we analyzed the competition between homomeri-zation of ToxR fusion proteins and their heterooligomerizationwith fusion proteins with the disabled ToxR DNA binding do-main (ToxR*; Fig. 3A). Disabled ToxR* fusion proteins caninteract with ToxR proteins in a dominant-negative fashion; theresulting decrease in RFP expression thus indicates the ratiobetween homooligomerization and heterooligomerization. Inthis assay, competition between homooligomer and hetero-oligomer formation with similar affinities results in a 50% de-crease in fluorescence (31).The Bax TMD has a strong tendency to self-associate (Figs. 1B

and 3B). As expected, this signal was attenuated by coexpressing

ToxR*/BaxTMD, whereas the ToxR*/GpATMD did not in-terfere with RFP expression (Fig. 3B). Interestingly, both TMDsderived from prosurvival Bcl-2 or Bcl-xL proteins interfered withBax TMD homooligomerization to at least the extent of the BaxTMD. These results suggest a strong capability of the TMDs ofprosurvival Bcl-2 proteins to heterooligomerize with the Bax TMD.Accordingly, the Bax TMD reduced Bcl-xL and Bcl-2 homo-oligomerization (Fig. 3 C and D). Strikingly, the Bcl-2 and Bcl-xLTMDs did not interfere with each other’s homooligomerization,suggesting an absence of heterooligomerization between both pro-survival Bcl-2 TMDs (Fig. 3 C and D). These results suggest that theinteractions of prosurvival Bcl-2 protein TMDs enable hetero-oligomerization with Bax. Interactions between the Bax TMD andTMDs of prosurvival Bcl-2 proteins could participate in Bax regu-lation, because the membrane-bound form of Bcl-xL has been de-scribed to insert only its TMD into the membrane (43). Therefore,the influence of TMD interactions on Bax regulation was tested byanalyzing isolated mitochondria permeabilization in HCT116 Bakknockout (KO) cells in the presence or absence of correspondingBax, Bcl-xL, Bcl-2, and Fis1 TMD segments. The presence of highBcl-xL or Bcl-2 TMD concentrations inhibited the release of Smacfrom mitochondria (Fig. 3E). These results show that hetero-oligomerization between Bax TMD and prosurvival Bcl-2 protein

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Fig. 2. Bax, Bcl-2, and Bcl-xL homooligomerize in the OMM. (A) BiFC assayanalyzing TMD homooligomerization by reconstitution of venus fluorescencefrom separate N and C termini fragments fused to TMD segments. (B) Bax,Bcl-2, and Bcl-xL TMD homooligomerization measured by BiFC in HCT116 cells.Fusions of b-Fos and b-Jun protein domains were used as positive controls (+),and the Δb-Fos/bJun pair served as a negative control (−); n = 3. Significantincreases compared with negative control were analyzed by using Dunnett’smultiple comparison test (95% confidence interval). Chimeric protein expres-sion of VN (c-myc) and VC (HA) constructs is compared in B, Lower; α-tubulinwas used as loading control. (C) Confocal images of HCT116 cells transfectedwith VC and VN constructs of the Bcl-2 and Bcl-xL, Bax, and Bax G197P TMDs.Formation of homooligomers (green and rainbow scale, first and second column,respectively) and mitochondria (red, third column), colocalized (yellow, fourthcolumn). (Scale bar, 20 μm.) (D) Self-association assays of wild-type and singleamino acid substitution variants of the Bax, Bcl-2, and Bcl-xL TMDs measured byBiFC in HCT116 cells. Error bars represent the mean ± SD, n ≥ 3. P valuesaccording to Dunnett’s test are displayed. *P < 0.05; **P < 0.01; ***P < 0.001. (E )Subcellular fractionation of HCT116 cells transfected with TMD constructswas controlled by using Tom20 (mitochondrial fraction; M) and α-tubulin(cytosol; C).

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Fig. 3. The Bax TMD interacts with antiapoptotic Bcl-xL and Bcl-2 TMDs.(A) Dominant-negative ToxR assay to analyze Bcl-2 protein TMD hetero-oligomerization. Coexpression of TMD constructs fused to wild-type ToxR(green) and TMD constructs fused to an inactive ToxR* mutant (yellow) inE. coli can result in heterooligomerization, leading to reduced RFP synthesis.(B) Effect of the Bax, Bcl-2, and Bcl-xL TMDs on ToxR/Bax TMD homo-oligomerization. The GpA TMD served as a control. (C) Effect of the Bax, Bcl-2,and Bcl-xL TMDs on Bcl-xL TMD homooligomerization as in B. (D) Effect of theBax, Bcl-2, and Bcl-xL TMDs on Bcl-2 TMD homooligomerization as in B. TheGpA TMD served as a control. (E) Smac release by endogenous Bax with andwithout tBid in the absence and presence of the Bax, Bcl-xL, Bcl-2, or Fis1 TMDpeptides from purified HCT116 Bak KO mitochondria. Smac was monitored inthe supernatant (S) and pellet (P) by Western blot. Bax and VDAC served ascontrols. Error bars represent the mean ± SD, n ≥ 3. P values according toDunnett’s test are displayed. *P < 0.05; **P < 0.01; ***P < 0.001.

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TMDs interferes with Bax-induced OMM permeabilization. How-ever, other mechanisms, such as competition for a common bindingsite or interference in the interaction with other OMM components,could also account for the observed effects. Bcl-xL TMD-dependentinhibition of OMMpermeabilization is not complete, because cyt c isstill released (Fig. S6). Lack of inhibition by the Bax TMD is par-ticularly interesting, because the peptide concentration exceeded thenative Bax protein concentrations. Therefore, symmetric Bax, andperhaps Bak oligomers, as recently suggested (27, 45), could toleratethe association of multiple TMDs. Alternatively, other oligomericBax structures could be more prominent in OMM permeabilization.

Bax TMD Interacts with Full-Length Proteins. Next, we tested thepotential of Bax, Bcl-2, and Bcl-xL TMDs to interact with full-length proteins. Self-associating VN/VC-BaxTMD or VN/VC-Bcl-2TMD chimeras that reconstitute the venus fluorescent proteinwere coexpressed with antiapoptotic and proapoptotic full-lengthproteins in HCT116 cells (Fig. 4A). BaxTMD homooligomeriza-tion was disturbed in the presence of full-length Bax, Bcl-2, andBcl-xL (Fig. 4B and Fig. S7A). Therefore, full-length proteins bindto the Bax TMD-derived chimeras, corroborating the interactions

between the isolated TMD segments (Fig. 3 B–D). Interestingly,Bax and Bcl-2 proteins interfered with Bcl-2 TMD homo-oligomerization, but Bcl-xL protein did not (Fig. 4C and Fig. S7B).The mitochondrial location of overexpressed TMD constructs wascorroborated by subcellular fractionation experiments (Fig. 4 Band C, Right). The specificity and TMD dependence of these in-teractions were tested by replacing the Bax TMD with the cor-responding Bcl-xL TMD segment in the full-length Bax protein(Bax/Bcl-xLtail). Bax/Bcl-xLtail interfered with Bax, but slightly al-tered Bcl-2 TMD homooligomerization (Fig. 4 B and C). On theother hand, the reciprocal chimera harboring the BaxTMD in full-length Bcl-xL (Bcl-xL/Baxtail) bound to Bax and Bcl-2 (Fig. 4 B andC). Then, the interaction between the TMDs of Bax and Bcl-2 andfull-length proteins is specific and depends on the TMD of theBcl-2 protein. These results were also corroborated in Bax/Bakdouble-KO (DKO) cells (Figs. S8 and S9).

Bax TMD Modulates Interactions with Endogenous Proteins andActivates Apoptosis. The Bcl-xL TMD is involved in Bax retro-translocation, and we thus focused our studies on the interactionsbetween both TMDs (13). A fusion of the Bax TMD to the Cterminus of the biotin ligase BirA (BirA/BaxTMD) biotinylatedendogenous Bax and Bcl-xL in nonapoptotic HCT116 cells (Fig. 5A and B). These results imply binding between Bax TMD andendogenous Bax and Bcl-xL proteins in the absence of apoptoticstimuli and independent of cytosolic (extramembranous) domain

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Fig. 4. Bcl-2 protein TMDs interact with full-length Bcl-2 proteins. (A) Heter-ooligomerization studies with the BiFC system using venus fluorescentprotein (VFP) reconstituted from VN/VC BaxTMD chimeras and full-length Bax(BaxFL). In this assay, a decrease in fluorescence indicates the formation ofheterooligomers between venus-derived chimeras and full-length Bcl-2proteins. (B) Oligomerization analysis of the Bax TMD in the presence of full-length Bax, Bcl-2, Bcl-xL, Bax-Bcl-xL tail, and Bcl-xL-Baxtail. VFP reconstitution byVN/VC BaxTMD chimeras was challenged with the indicated full-length proteinsin HCT116 cells (B, Left). Subcellular fractionation showed mitochondrial (M)localization of VN/Bax and VC/Bax in the absence or presence of full-lengthproteins. C indicates the cytosolic fraction. (C) Analysis of Bcl-2 TMD oligomeri-zation in the presence of full-length Bax, Bcl-2, Bcl-xL, Bax–Bcl-xL tail, and Bcl-xL–Baxtail proteins in HCT116 cells. Subcellular fractionation showed Bcl-2 TMD-derived constructs localization in the absence or the presence of full-lengthproteins. Tom20 and α-tubulin served as controls. *P < 0.05; ***P < 0.001.

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wt DH5 DH6 wt DH5DH6

Fig. 5. Bax TMD interacts with endogenous Bax and Bcl-xL. (A) The BirA in situproximal biotinylation assay was used to identify interactions between the BaxTMD and endogenous Bax and Bcl-xL proteins in human cells. After the ex-pression of BirA/BaxTMD, proteins interacting with Bax TMD (blue) were labeledwith biotin (star) by BirA. Crude cell extract was subsequently applied to a biotinaffinity matrix (solid line). Bound (labeled) proteins were analyzed by SDS/PAGEand Western blot. (B) Input and labeled proteins of the biotin affinity matrixfrom HCT116 cells expressing BirA/BaxTMD fusion were analyzed by West-ern blot. Tom20 served as a control. (C) BirA fusion assay analyzing BirA/BaxFL-interactions with wild-type Bax compared with Bax ΔH5 and Bax ΔH6 presentin the labeled fraction (L) compared with the input (I) by Western blot. n = 3.(D) BirA fusion assay comparing BirA/BaxFL and BirA/ BaxTMD interactions witheither endogenous Bcl-xL (Bcl-xL WT) or ectopically expressed wild-type myc-Bcl-xL or themyc-Bcl-xL G138A variant byWestern blot. Tom20 serves as control. n= 3.(E) The BirA fusion assay was used to identify interactions between Bax TMDmutants and endogenous Bax and Bcl-xL proteins in human cells.

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interactions. Our results are supported by Bak (and Bax) TMD in-terfaces mediating homooligomerization in the absence of BH3-dependent interactions (45). Mutant Bax proteins lacking eitheralpha 5 (Δ5) or alpha 6 (Δ6) helices, which are potentially relevantfor membrane insertion and pore formation (19, 26, 28, 46), retainedthe capacity for Bax association in mitochondrial membranes (Fig.5C), emphasizing the role of the TMDs in early protein interactions.Both BirA/BaxFL and BirA/BaxTMD bound to wild-type Bcl-xL (Fig.5D). This binding was strongly reduced when cells were cotransfectedwith the nonfunctional Bcl-xL G138A mutant (47). Strikingly, somecapacity for heterooligomer formation was retained (Fig. 5D) andmatches the Bax TMD capability of oligomer formation, corrobo-rating the contribution of Bax TMD interactions with prosurvivalBcl-xL. Mutations in the BirA/BaxTMD construct that decrease Baxhomooligomerization, such as G179P, provoked a similar decrease inBcl-xL biotinylation (Fig. 5E), suggesting at least a partial commoninterface for heterooligomerization and homooligomerization.Together, our results demonstrate that interactions between the

TMDs of Bax and Bcl-xL occur in the OMM of human cells beforethe induction of apoptosis. The analysis of a potential role ofTMD interactions in apoptosis induction revealed that the ectopicexpression of the BaxTMD in HCT116 cells induces caspase-3/7activation (Fig. 6). BaxTMD G179P, but not BaxTMD F176A,interferes with this activation, in good agreement with the impactof these mutants on oligomerization in Fig. 2D. Furthermore,cotransfection with antiapoptotic Bcl-2- and Bcl-xL TMD-derivedconstructs or with full-length proteins significantly reduced apo-ptosis activation (Fig. 6). Therefore, Bcl-2 protein TMD interac-tions are involved in mitochondrial apoptosis signaling.

ConclusionAntiapoptotic and proapoptotic Bcl-2 proteins regulate mitochon-drial apoptosis signaling, and thus the cell fate, by dynamic interac-tions. Interplay between the BH3 domains and hydrophobic groovesof the respective interaction partner have been characterized (48). In

the present study, we discovered interactions between the TMDs ofBax, Bcl-2, and Bcl-xL that occur in nonapoptotic cells and modu-late mitochondrial apoptosis signaling. The consistent picture thatemerges from these studies is that Bcl-2 and Bcl-xL TMDs couldhave the ability to regulate Bax pore-forming activity by means ofdirect competition, leading to the formation of heterooligomersthat abate Bax homooligomer formation and OMM per-meabilization. The existence of Bax TMD interactions has beenproposed based on cross-linking experiments (49, 50) and 3Dmodels (28) and has been suggested to contribute to the en-largement of the Bax pore (51). Although not as tight as hy-drophobic groove and BH3 domain interactions, TMD-TMDinteractions are sufficient for heterooligomerization and homo-oligomerization of Bcl-2 proteins. Therefore, Bax dimers andoligomers could facilitate lateral sorting in the OMM or theformation of Bcl-2 protein-containing complexes (52). TMD–

TMD interactions are consistent with models of Bax activation,suggesting separation of helices α5 and α6 (27, 53) and theconcerted insertion of both helices into the OMM (51, 54).Conversely, Bax activation according to the clamp model wouldrequire a sequential mechanism to allow formation of antipar-allel TMD interactions (28). The observation of TMD–TMDinteractions between Bcl-2 proteins in proliferating cells furtheremphasizes the necessity to assess the protein conformation ofBax and other Bcl-2 proteins. These new surfaces of protein–protein interaction among proapoptotic and prosurvival mem-bers could represent attractive targets for selective drug design.

MethodsMethods are fully described in SI Methods. ToxRed chimeric constructs weregenerated by specific primer annealing of Bcl-2 protein TMDs in the HindIII/XhoI restriction sites of ToxRed vectors (Table 1 and Table S1). The maltosecomplementation assay was performed as described (39, 55).

ToxRed Oligomerization Assays. ToxR–Bcl-2 TMD constructs (Table S1) weretransformed into the malE mutant E. coli MM39 strain. For RFP measurements,24-well plates were adjusted to equivalent growth (OD600 0.6–0.8), and theRFP emission spectra were collected by a Wallac 1420 Workstation (ʎexc 560and ʎem 595 nm).

BiFC-TMD Assays. BiFC assays were performed as described (56). An improvedBiFC assay with a high signal-to-noise ratio was selected to avoid back-ground interference (57, 58). The system was adapted to clone Bax, Bcl-xL,and Bcl-2 TMDs at the C terminus of venus protein fragments, according totheir natural topology in full-length proteins.

BirA Interaction Partner Identification. HCT116 Bax/Bak DKO cells were trans-fected with pcDNA3-mycBioID-Bax plasmid, resulting in the expression of myc-tagged BirA/Bax fusion. The cell lysate was incubated with streptavidinagarose beads (Thermo) at 4 °C overnight. Input and bead samples wereresolved on a 10% (wt/vol) SDS/PAGE and analyzed by Western blot for theindicated proteins.

ACKNOWLEDGMENTS. We thank Prof. W. DeGrado (University of California,San Francisco School of Pharmacy) for kindly providing the ToxR plasmids; Profs.R. Youle and B. Vogelstein for kindly providing the human colorectal carcinomaHCT116 cells; Prof. D. Langosch for critical reading of the manuscript and helpfulsuggestions; and Maria J. Garcia-Murria, Sylvia Liebscher, and Alicia García-Jareño for technical assistance. This work was supported by Spanish Ministryof Economy and Competitiveness Grants BFU2016-79487, SAF2014-52614-R, andBEFPI/2013/A/046; Generalitat Valenciana Grant PROMETEOII/2014/061; the EmmyNoether program; the Heisenberg program and the Sonderforschungsbereich746 of the German Research Council (Deutsche Forschungsgemeinschaft); andthe Centre for Biological Signalling Studies (BIOSS, EXC-294) funded bythe Excellence Initiative of the German Federal and State Governments.V.A.-F. and E.L. were supported by Generalitat Valenciana Grants BEFPI/2013/A/046 and ACIF/2016/019.

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FLL

casp

ase

3/7

activ

ity (R

FU) VN-BaxTMD

**

Bcl-x

FL

Bcl-2

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pe

LNT

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Bcl-2 T

MD

BaxTMD G

179P

BaxTMD F17

6A0

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*** ***

10000

Fig. 6. TMD interactions modulate apoptotic response. Effect of VN/BaxTMD variants, VN/Bcl-2TMD and VN/Bcl-xLTMD chimeric proteins, or Bcl-2and Bcl-xL full-length proteins on caspase-3/7 activity induced by VN-BaxTMDin HCT116 cells is shown. Caspase 3/7 activity was analyzed in cytosolic ex-tracts 24 h after transfection. Error bars represent the mean ± SD, n = 3. Pvalues according to Dunnett’s test are displayed. **P < 0.01; ***P < 0.001.

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